1: Determine the mechanisms of biofilm formation in foodborne pathogens and elucidate the role of biofilms in persistence of pathogens in food environments. 1.1 Assemble and screen a collection of Shiga toxin-containing Escherichia coli (STEC) for biofilm forming properties. 1.2 Molecular characterization of biofilm formation in non-O157 STEC. 1.3 Identification of novel factors necessary for biofilm formation in non- O157 STEC. 1.4 Mixed biofilm formation between STEC and isolates from food processing environments.
2: Examine the role of quorum sensing of microorganisms in food environments, with specific emphasis on quorum sensing in mixed biofilm formation and the role of autoinducers such as AHL in survival. 2.1 Examine the role of quorum sensing in biofilm formation by non-O157 STEC.
3: Examine the persistence and transmission of antimicrobial resistant bacteria in microbial ecosystems, with specific emphasis on mobilizable plasmids carrying antibiotic resistance genes. Specifically, conduct sequence analyses and determine phylogenetic relationships among mobilizable plasmids carrying genes encoding antibiotic resistance and investigate gene transfer in biofilms. 3.1 Examine the prevalence and persistence of the KanR ColE1-like plasmids in Salmonella serovars isolated from sick animals and their environmentâ€“ a longitudinal study. 3.2 Investigate plasmid transmission and persistence in biofilms using the KanR ColE1-like mobilizable plasmids as model systems.
4: Qualitatively and quantitatively characterize microbial communities associated with food and food processing environments and examine the role of predominant species in pathogen persistence in mixed culture biofilms. 4.1 Develop a DNA-based most probable composition protocol for estimating the total number, as well as the type, of organisms in an environmental sample or biofilm. 4.2 Determine relative concentrations of various foodborne organisms. Sampling from select food or processing locales, culturable isolate plating & selection, PCR amplification, gene cloning, plating and selection.
Approach: Microbes rarely exist in the environment as a monoculture but are present in complex microbial communities. Microorganisms in these communities often engage in a wide range of intercellular behaviors that may affect the presence and persistence of pathogens in foods. The primary aims of this project are to gain a better understanding of some of the complex social behaviors of foodborne pathogens and to catalog bacterial communities associated with selected foods and food processing environments. These aims will be accomplished by: 1) studying the genetic factors contributing to biofilm formation in Shiga toxin-producing E. coli (STEC) and the role of cell-to-cell communication (quorum sensing) in biofilm formation in STEC, 2) studying the potential for mixed biofilm formation between STEC and non-pathogenic environmental flora, 3) examining the prevalence and persistence of antimicrobial resistance plasmids in Salmonella strains isolated from natural environments and investigating the transmission and persistence of these plasmids in model biofilms composed of Salmonella and/or STEC, and 4) developing and applying sampling and sequencing methods to qualitatively and quantitatively determine the members of microbial communities in beef products and beef processing facilities. Collaborations with ARS, corporate, and university partners have been established to ensure that all aspects of the work can be accomplished.
Inactivation of avirulent pgm+ and Δpgm Yersinia pestis by ultraviolet light (UV-C)
Paoli GC, Sommers CH, Scullen OJ, Wijey C.
Food Microbiol. 2014 Dec;44:168-72.
Variability in DNA polymerase efficiency: effects of random error, DNA extraction method, and isolate type
Irwin P, Nguyen LH, Chen CY, He Y.
J of Stat and Math. 2014 Aug 22;1(1):1-16.
Automated immunomagnetic separation for the detection of Escherichia coli O157:H7 from spinach
Chen J, Shi X, Gehring AG, Paoli GC.
Int J Food Microbiol. 2014 Jun 2;179:33-7.
Growth media and temperature effects on biofilm formation by serotype O157:H7 and non-O157 Shiga toxin-producing Escherichia coli
Uhlich GA, Chen CY, Cottrell BJ, Nguyen LH.
FEMS Microbiol Lett. 2014 May;354(2):133-41.
Phenotypic and genotypic antimicrobial resistance traits of foodborne Staphylococcus aureus isolates from Shanghai
Xu J, Shi C, Song M, Xu X, Yang P, Paoli G, Shi X.
J Food Sci. 2014 Apr;79(4):M635-42.
Phenotypic and genotypic characterization of biofilm forming capability in non-O157 Shiga toxin-producing Escherichia coli strains
Chen C, Hofmann CS, Cottrell BJ, Strobaugh Jr TP, Paoli G, Nguyen LT, Yan X, Uhlich GA
PLoS One. 2013 Dec 27;8(12):e84863.
Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157:H7
Uhlich GA, Chen C, Cottrell BJ, Hofmann CS, Dudley E, Strobaugh Jr TP, Nguyen LT.
Microbiology. 2013 Aug;159(8):1586-96.
A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction
Bai Y, Song M, Cui Y, Shi C, Wang D, Paoli GC, Shi X.
Anal Chim Acta. 2013 Jul 17;787:93-101.
Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products
Irwin PL, Reed SA, Brewster JD, Nguyen LT, He Y.
Anal Bioanal Chem. 2013 Mar;405(7):2353-69.
Peroxide resistance in Escherichia coli serotype O157 : H7 biofilms is regulated by both RpoS-dependent and -independent mechanisms
Uhlich GA, Chen CY, Cottrell BJ, Irwin PL, Phillips JG.
Microbiology. 2012 Sep;158(Pt 9):2225-34.
Evaluation of Fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for sub-typing Escherichia coli O157:H7 isolates
Davis R, Paoli G, Mauer LJ.
Food Microbiol. 2012 Sep;31(2):181-90.
A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration
Uhlich GA, Chen CY.
Plasmid. 2012 May;67(3):259-63.
A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's
Irwin PL, Nguyen LH, Chen CY, Uhlich GA, Paoli GC.
Anal Bioanal Chem. 2012 Mar;402(9):2713-25.
Genetically marked strains of Shiga toxin-producing E. coli O157:H7 and non-O157 for detection and modeling - (Abstract Only)
Production and regulation of functional amyloid curli fimbriae by Shiga toxin-producing Escherichia coli - (Abstract Only)
Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette - (Abstract Only)
Applegate, B., Rosenfield, C., Martinez, M., Zhu, F., Farrokhzad, K., Paoli, G. 2014. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette. Meeting Abstract. 1-2.
Media composition and incubation temperature affect Congo red dye affinity of Shiga toxin-producing Escherichia coli - (Abstract Only)
Uhlich, G.A., Chen, C., Cottrell, B.J., Nguyen, L.T. 2014. Media composition and incubation temperature affect Congo red dye affinity of Shiga toxin-producing Escherichia coli. Meeting Abstract. MA.
Characterization and distribution of ColE1-like kanamycin-resistance plasmids in Salmonella enterica from food animals - (Abstract Only)
Chen, C., Strobaugh Jr, T.P., Nguyen, L.T., Abley, M., Lindsey, R.L., Cray, P.J. 2014. Characterization and distribution of ColE1-like kanamycin-resistance plasmids in Salmonella enterica from food animals. Meeting Abstract.
Prophage insertions in mlrA are not the major obstacle to biofilm formation in Escherichia coli O157:H7 - (Abstract Only)
Uhlich, G.A., Chen, C., Dudley, E.G., Hofmann, C.S., Cottrell, B.J. 2014. Prophage insertions in mlrA are not the major obstacle to biofilm formation in Escherichia coli O157:H7. Meeting Abstract. MA.
Mutations in the rpoS gene are the major limiting factor for biofilm formation in Escherichia coli serotype O157:H7 clinical isolates - (Abstract Only)
Chen, C., Uhlich, G.A., Strobaugh Jr, T.P., Cottrell, B.J., Dudley, E.G., Hofmann, C.S., Nguyen, L.T. 2013. Mutations in the rpoS gene are the major limiting factor for biofilm formation in Escherichia coli serotype O157:H7 clinical isolates. Meeting Abstract. MA.
Nonpeptidic mimics of host defense proteins as antimicrobial agents for E. coli O104:H4, campylobacter spp. and other foodborne pathogens - (Abstract Only)
Mithal, S., Chen, C., Scott, R. 2012. Nonpeptidic mimics of host defense proteins as antimicrobial agents for E. coli O104:H4, campylobacter spp. and other foodborne pathogens. Meeting Abstract. Poster F-1494.
Understanding the role of the catalase/peroxide genes in H2O2 resistance of E. coli serotype O157:H7 biofilms - (Abstract Only)
Uhlich, G.A., Chen, C., Irwin, P.L. 2012. Understanding the role of the catalase/peroxide genes in H2O2 resistance of E. coli serotype O157:H7 biofilms [abstract]. 1:1.
Regulation of the csgD promoter by global regulators HN-S, IHF, and RpoS in E. coli O157:H7 isolates - (Abstract Only)
Chen, C., Uhlich, G.A. 2012. Regulation of the csgD promoter by global regulators HN-S, IHF, and RpoS in E. coli O157:H7 isolates [abstract]. Poster p-3-121.